Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: KEGG:D00046 (lactose)
16,692 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Escherichia coli K-12 mutants that are resistant to bacteriophage chi, defective in motility, and unable to grow at high temperature (42 degrees C) were isolated from among those selected for rifampin resistance at low temperature (30 degrees C) after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Genetic analysis of one such mutant indicated the presence of two mutations that probably affect the beta subunit of ribonucleic acid (RNA) polymerase: one (rif) causing rifampin resistance and the other (Ts-74) conferring resistance to phage chi (and loss of motility) and temperature sensitivity for growth. Observations with an electron microscope revealed that the number of flagella per mutant cell was significantly reduced, suggesting that the Ts-74 mutation somehow affected flagella formation at the permissive temperature. When a mutant culture was transferred from 30 to 42 degrees C, deoxyribonucleic acid synthesis accelerated normally, but RNA or protein synthesis was enhanced relatively little. The rate of synthesis of beta and beta' subunits of RNA polymerase was low even at 30 degrees C and was further reduced at 42 degrees C, in contrast to the parental wild-type strain. Expression of the lactose and other sugar fermentation operons, as well as lysogenization with phage lambda, occurred normally at 30 degrees C, suggesting that the mutation does not cause general shut-off of gene expression regulated by cyclic adenosine 3',5'-monophosphate.
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PMID:Ribonucleic acid polymerase mutant of Escherichia coli defective in flagella formation. 19 75

The antibiotic rifampicin inhibits transcription initiation, but not the elongation and completion of nascent RNA transcripts. Addition of low concentrations of rifampicin only partially blocks initiation but at the same time specifically alters the general pattern of transcription in the culture. The transcription of genes specifying the beta and beta' subunits of RNA polymerase, and to a lesser extent of the genes specifying the RNA and protein components of the ribosome, was specifically stimulated relative to total transcription. In contrast, the transcription of the lactose operon was selectively reduced. These results are consistent with the ideas that the level of expression of the genes specifying the beta and beta' subunits is sensitive to the general rate of RNA synthesis in the culture, and that the expression of the beta and beta' RNA polymerase genes is related to the expression of ribosome component genes.
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PMID:Gene expression in Escherichia coli B/r during partial rifampicin-mediated restrictions of transcription initiation. 36 68

The chemical alkylating agent dimethyl sulfate can probe the interaction between Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) and the purine bases of a promoter. This agent methylates the N7 position on guanine or the N3 position on adenine; the bound protein can either protect these positions or affect the reactivity to produce an enhanced methylation. The pattern of DNA residues in the lactose promoter protected from, or enhanced to, methylation by a specifically bound polymerase shows that the enzyme covers a region of at least 38 base pairs, stretching upstream from the origin of transcription. These protein-DNA contacts occur predominantly in the major groove of the DNA helix. Furthermore, this pattern of methylation shows that the polymerase unwinds the helix at the origin of transcription. The relationship between polymerase-DNA contacts defined by dimethyl sulfate and known features of promoter structure is discussed. To facilitate these experiments I have constructed a plasmid that permits a unique 5'-end labeling of each strand of a 95-base-pair fragment containing a lac operon promoter. This plasmid contains two copies of the lac promoter-operator region.
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PMID:Contacts between Escherichia coli RNA polymerase and a lac operon promoter. 36 74

The L-arabinose operon in Escherichia coli is a model system for the study of the control of gene expression. Maximal expression of the araBAD operon requires two positive control components: the araC protein-L-arabinose complex and the cyclic AMP receptor protein-cyclic AMP complex. Both araC protein and cyclic AMP receptor protein are required for the initiation of transcription of araBAD mRNA. We have used the plasmid pBR322 as a vector for cloning DNA fragments that contain the araBAD promoter. The cloned ara fragments were identified by both physical and genetic tests. A restriction map was constructed and the DNA sequence of the promoter was determined. The promoter contains a site that is similar to the RNA polymerase recognition sites in the galactose and lactose operons. It also contains a region similar to the known cyclic AMP receptor protein binding sites in the galactose and lactose operons.
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PMID:DNA sequence of the araBAD promoter in Escherichia coli B/r. 36 97

Theoretical calculations were made to determine the influence of side specific 'melting' and 'stabilizing' proteins on the thermal stability of nearby base pairs (bp). A DNA sequence 999bp. long containing the 123 bp. lactose operon control region in the center was examined. Melting curves of base pairs near the binding sites of the catabolite activator protein, CAP, the lactose repressor, and RNA polymerase were calculated in the absence and presence of each protein. The empirical loop entropy model of the helix-coil transition of DNA was employed. Calculations show that melting and stabilizing proteins alter the tm of base pairs 20 to 100 bp-away. The magnitude and range of the effect is strongly influenced by the base pair composition and sequence of the protein site and the immediately adjacent DNA regions.
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PMID:The transmission of stability or instability from site specific protein-DNA complexes. 90 92

Incubation of UV-irradiated plasmid DNA with a protein extract prepared from Escherichia coli cells led to the production of mutations in the cro gene residing on the plasmid. The mutations were detected in a subsequent bioassay step, which involved transformation of an indicator strain with the plasmid DNA that was retrieved from the reaction mixture, followed by plating on lactose/MacConkey plates. UV mutations produced in this cell-free reaction required the recA and umuC gene products and were prevented by rifampicin, an inhibitor of RNA polymerase, which inhibited plasmid replication. Removal of pyrimidine photodimers from the plasmid by enzymatic photoreactivation after the in vitro stage, but prior to transformation, increased plasmid survival as expected. Surprisingly, it also caused a large increase in the frequency of UV mutations detected in the bioassay. This photoreactivation-stimulated in vitro UV mutagenesis was dependent on the excision repair genes uvrA, uvrB, and uvrC and occurred in the absence of DNA replication. This suggests that two distinct UV mutagenesis pathways occurred in vitro: a replication-dependent pathway (type I) and a repair-dependent pathway (type II). DNA sequence analysis of type II UV mutations revealed a spectrum similar to that of in vivo UV mutagenesis. When the photoreactivation step was included in the protocol, type II UV mutagenesis did not require the RecA and UmuC proteins. These results are in agreement with the in vivo delayed photoreactivation phenomenon, where the removal of photodimers after an incubation period eliminated the requirement for RecA and UmuC in UV mutagenesis. The above system will enable the biochemical analysis of UV mutagenesis and the isolation of proteins involved in the process.
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PMID:Biochemical analysis of UV mutagenesis in Escherichia coli by using a cell-free reaction coupled to a bioassay: identification of a DNA repair-dependent, replication-independent pathway. 131 85

Cooperative interactions between regulatory proteins and RNA polymerase are a common feature of transcriptional systems. We have developed a method, based on the electrophoresis mobility shift assay, for the measurement of cooperative effects in the binding of proteins to DNA restriction fragments. Using this approach we have identified a hitherto unknown interaction between the E. coli lactose repressor and CAP proteins. We suggest that this interaction plays a role in the control of the lactose operon that is not predicted by current regulatory models.
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PMID:Cooperative interactions in transcriptional regulation. 136 5

The lactose promoter-operator region of Escherichia coli contains two binding sites for cyclic AMP receptor protein (CAP), two for the lactose repressor, and two for RNA polymerase. The high density of binding sites makes cooperative interactions between these proteins likely. In this study, we used the gel electrophoresis mobility shift assay and binding partition analysis techniques to determine whether the secondary CAP site influences the binding of CAP to the principal CAP site in the lactose promoter when both are present on a linear DNA molecule. Such an effect could occur through the formation of a bridged DNA-CAP-DNA structure, through the interaction of CAP molecules bound to each of the sites, or through allosteric effects caused by CAP-mediated DNA bending. We found, however, that the interaction of CAP with these sites was not cooperative, indicating that CAP sites 1 and 2 bind CAP in an independent manner.
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PMID:The binding of cyclic AMP receptor protein to two lactose promoter sites is not cooperative in vitro. 198 34

Urea, at concentrations which do not interfere with bacterial growth, specifically inhibits the expression of catabolite sensitive operons. To search for the target and the mechanism of urea action we measured lactose (lac) and tryptophanase (tna) specific mRNA synthesis in vivo and in vitro. We show that urea acts by two different mechanisms at these two catabolite sensitive operons, resembling the manner in which catabolite repression regulates lac and tna. At the lac promoter, urea abolishes transcription initiation or blocks an early step in mRNA elongation without interfering with the binding of RNA polymerase and catabolite gene activator protein (CAP). At the tna promoter, urea does not abolish transcription initiation but could interfere with tnaC translation.
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PMID:Two different mechanisms for urea action at the LAC and TNA operons in Escherichia coli. 216 52

A series of deletion mutants of the wild-type Escherichia coli lactose promoter, with endpoints at +25, +19, +14, +1 and -6 (relative to the start of transcription at +1), was constructed and the deleted DNA replaced with non-lac DNA. These mutants were used to show that no specific DNA sequences downstream from -6 are required for efficient promoter utilization in vitro. In all cases transcription is dependent on the presence of the catabolite activator protein (CAP) and cAMP, and begins at +1 at a level indistinguishable from that at the wild-type promoter. A set of lac DNA fragments deleted to -6 was constructed, having an A, C, G or T residue at +1 and heterologous DNA downstream. These synthetic promoters allow systematic testing of the effect of the initiating nucleotide on the transcription process. Again, transcription occurs mainly from +1, at a level similar to the normal wild-type level. No substantial differences between these promoters are observed in the rates of formation of stable complexes, in the degree of complex formation, in the rate at which polymerase "escapes" from the complex or in abortive transcription products. Equivalent results are seen with a related set of constructs based on the CAP-insensitive lac UV5 promoter. Thus, lac promoter sequences including consensus hexamers at -10 and -35, plus the spacer region between them, provide specificity and efficiency both in initiation of transcription by RNA polymerase and in CAP-polymerase interactions. A question as to whether there is a third RNA polymerase binding site at lac, in addition to the known overlapping P1 and P2 regions, was not unambiguously answered. However, if a "P3" site does exist, it must lie between P1 and P2. Alternatively, the variety of polymerase interactions at wild-type lac may reflect different structural states of the enzyme. The results presented here indicate that DNA downstream from -6 plays little part in determining the conformation of the enzyme at the lactose promoter.
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PMID:Specific sequences downstream from -6 are not essential for proper and efficient in vitro utilization of the Escherichia coli lactose promoter. 225 29


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